EP2073004A1 - Analyse électrophorétique de la mitochondrie isolée pour la détection des dommages cellulaires ou tissulaires - Google Patents

Analyse électrophorétique de la mitochondrie isolée pour la détection des dommages cellulaires ou tissulaires Download PDF

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EP2073004A1
EP2073004A1 EP07024623A EP07024623A EP2073004A1 EP 2073004 A1 EP2073004 A1 EP 2073004A1 EP 07024623 A EP07024623 A EP 07024623A EP 07024623 A EP07024623 A EP 07024623A EP 2073004 A1 EP2073004 A1 EP 2073004A1
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Prior art keywords
mitochondria
mitochondrial
sample
populations
surface charge
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Hans Zischka
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Helmholtz Zentrum Muenchen Deutsches Forschungszentrum fuer Gesundheit und Umwelt GmbH
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Priority to EP07024623A priority Critical patent/EP2073004A1/fr
Priority to PCT/EP2008/010915 priority patent/WO2009077205A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G01N33/5079—Mitochondria

Definitions

  • the present invention relates to a method for detecting mitochondrial damage in mammalian tissue or cells comprising: (a) isolating mitochondria from a sample obtained from a mammal and containing tissue or cells containing mitochondria; (b1) separating populations of the isolated mitochondria of step (a) by means of their electrophoretic mobility; and (c1) comparing the populations of the mitochondria obtained in step (b1) with those of a reference sample containing tissue or cells displaying a non-pathological phenotype, wherein a rise in the number of populations, a shift of the population(s) towards the anode or the cathode and/or an increase in the heterogeneity of one or more populations separated in step (b1) as compared to those of the reference sample is indicative of mitochondrial damage; or (b2) analyzing the average surface charge of the isolated mitochondria of step (a); and (c2) comparing the average surface charge determined in step (b2) with that of a reference sample containing cells displaying a non-pathological phenotype, wherein a shift of
  • Mitochondria are the powerhouse of the cell and linked to many fundamental cellular processes. They are involved in many metabolic and anabolic processes and play an important role in cell death in higher eukaryotic life. Low mitochondrial production of reactive oxygen species (ROS) has been linked to longevity of the cell. Cell fate in terms of apoptosis or/and necrosis is frequently determined by one crucial event: mitochondrial outer membrane permeabilization (MOMP). MOMP marks the "point of no return" for the cell, after which it is committed to death. MOMP leads to the release of proteins normally found in the space between the inner and outer mitochondrial membranes (e.g. cytochrome c and AIF).
  • ROS reactive oxygen species
  • MOMP may be induced directly or may be the culmination of a sequence of events starting with an increase in the permeability of the mitochondrial inner membrane called permeability transition (PT).
  • PT was shown to be unspecifically induced e.g. by calcium (Hunter at al., 1976). Since then a plethora of studies have dealt with this phenomenon which investigated the mechanistic, molecular and pharmacological aspects of PT. It was shown that PT could be inhibited by submicromolar concentrations of the immunosuppressant drug cyclosporine A and that it involves the creation of a pore/channel-like structure (Crompton et al., 1988; Sorgato et al., 1987; Szabo and Zoratti, 1992).
  • Factors that cause the PT pore to close or remain closed include acidic conditions (Friberg and Wieloch, 2002), high concentrations of ADP (Brustovetsky et al., 2003; Hunter and Hayworth 1979), high concentrations of ATP (Beutner et al., 1998), and high concentrations of NADH (Hunter and Hayworth, 1979). Divalent cations like Mg 2+ also inhibit PT because they can compete with Ca 2+ for the Ca 2+ binding sites on the matrix side of the PT pore (Hayworth and Hunter, 1979). Whether the mitochondria resulting from the above PT-inducing stimuli are all equally damaged or whether different stages of damage exist is poorly established.
  • the present invention relates to a method for detecting mitochondrial damage in mammalian tissue or cells comprising: (a) isolating mitochondria from a sample obtained from a mammal and containing tissue or cells containing mitochondria; (b1) separating populations of the isolated mitochondria of step (a) by means of their electrophoretic mobility; and (c1) comparing the populations of the mitochondria obtained in step (b1) with those of a reference sample containing tissue or cells displaying a non-pathological phenotype, wherein a rise in the number of populations, a shift of the population(s) towards the anode or the cathode and/or an increase in the heterogeneity of one or more populations separated in step (b1) as compared to those of the reference sample is indicative of mitochondrial damage; or (b2) analyzing the average surface charge of the isolated mitochondria of step (a); and (c2) comparing the average surface charge determined in step (b2) with that of a reference sample containing cells displaying a non-pathological phenotype
  • Mitochondrial damage refers to the mitochondrial outer membrane permeabilization (MOMP).
  • MOMP is either the result of an increase in the permeability of the mitochondrial inner membrane which is called permeability transition (PT). This increase can occur to different extents leading either to reversible mitochondrial damage where only PT is present or to MOMP, the latter comprising different degrees of membrane rupture and mostly leading to cell death.
  • the outer mitochondrial membrane may be targeted and permeabilized directly by a variety of different stimuli (e.g. the protein BAX).
  • the extent of mitochondrial damage is defined by the extent of the mitochondrial inner membrane exposed which may range from minor ruptures or pores (exposure of less than 1 to 10% of the inner membrane) via intermediate rupture (exposure of more than 10%, such as 20, 30 or 40% to up to 50% of the inner membrane) to an almost complete removal of the outer membrane (exposure of more than 50%, such as 60, 70, 80 or 90% to 100% of the inner membrane).
  • MOMP mitochondrial outer membrane permeabilization
  • Isolating mitochondria as used in connection with the present invention defines the step of obtaining mitochondria to be applied to the separation/analysis step of the method of the present invention from a sample. By isolating the mitochondria, they are separated from the remainder of the cell so that their surface is not covered by cell debris such as fragments of the cell membrane but is exposed and accessible for external influences such as electric fields.
  • the skilled person is aware of methods for isolating mitochondria (for exemplary methods see Pallotti and Lenaz, 2007). An exemplary method is described in Example 1 below.
  • the term "electrophoretic mobility" determines the extent or velocity of movement of a charged particle in a given electric field. It depends on several physical parameters, in particular on the surface charge, but also on the size of the particles.
  • sample denotes a sample from a human, a rodent, a ruminant or any mammal commonly used in laboratory experiments.
  • a sample can be liquid or solid and contains cells from a tissue or organ.
  • a sample to be analyzed has undergone or is at least suspected of having undergone mitochondrial damage and has a pathological phenotype.
  • a pathological phenotype is characterized in that it exhibits symptoms of a pathological condition caused e.g. by unusual stress such as oxidative stress, inflammation or infection.
  • the term “populations of mitochondria”, interchangeably used with “subpopulations of mitochondria” denotes groups of mitochondria which differ by their electrophoretic mobility or surface charge or both. Depending on the extent of permeability transition or MOMP which the mitochondria undergo, these properties change resulting in populations or subpopulations of mitochondria to which a certain degree of damage can be assigned.
  • reference sample denotes a sample from the same or a different individual as the one from which the sample to be analyzed was taken. If a sample from the same individual is used as a reference sample, it can be taken from an undamaged part of the same tissue or organ as the sample to be measured or from a different tissue or organ with a reaction in response to stimuli which is comparable to that of the tissues or organs the sample was taken from and/or with comparable properties as regards susceptibility to substances causing mitochondrial damage. Alternatively, the reference sample can be taken from the same individual from the same tissue or organ before the damage happens, resulting in a direct comparison of the distribution of mitochondrial subpopulations before and after the damage.
  • a sample of the organ(s) or tissue(s) to be treated or otherwise in danger of being harmed can be taken prior to the operation and another one after the operation.
  • HMM heart-lung-machine
  • the method of the present invention can be used to determine mitochondrial damage caused following ischemia/reperfusion (ischemia/reperfusion disease).
  • the reference sample can be taken from a different individual.
  • the reference sample is preferably taken from the same tissue or organ as the sample to be analyzed.
  • the individual is preferably from the same species as that from which the sample to be measured was obtained.
  • the reference sample does not necessarily have to be prepared simultaneously to the sample. It may well be stored under preserving conditions, e.g. frozen, prior to the sample analysis. In the case of analyzing the average surface charge, the reference sample may well be separated and/or analyzed prior to the sample analysis and the results of the analysis may be stored electronically or otherwise and be retrievable upon request.
  • a "non-pathological phenotype" in the context of the present invention relates to the apparent state of the cells of the reference sample as normal, i.e. they display a phenotype which is to be expected from a sample which is not affected by a pathological condition, in particular has not undergone any unusual stress such as oxidative stress, inflammation, infection or is concerned with other diseases resulting in an altered metabolism or an altered state of the mitochondria, in particular an altered mitochondrial metabolism or mitochondrial damage.
  • zone electrophoresis in a free flow device could be useful for the preparative analysis of yeast mitochondria (Zischka et al., 2006) which differ significantly from mammalian mitochondria in terms of (protein-) composition, biological functions and organization. Furthermore, important differences were found between the electrophoretic deflection behaviour of yeast mitochondria and mitochondria isolated from mammalian systems. Based on the current knowledge, electrophoretic deflection of mammalian mitochondria is primarily dependent on the surface charge whereas the electrophoretic deflection of yeast mitochondria is dependent on several other aspects like hydrodynamic properties. Importantly, the study of the electrophoretic deflection of yeast mitochondria was not remotely related to PT or PT-induced MOMP or a disease causing any of the two and is thus not related to the present invention.
  • the inner surface of rat liver mitochondria is more negatively charged than the surface of their enveloping outer membranes.
  • MOMP the total surface charge of mitochondria is altered due to the simultaneous exposure of the differently charged outer and inner mitochondrial membranes. Accordingly, by detecting differences in the total charges of mitochondria due to the additional exposure of inner charges, the method of the present invention can detect not only whether mitochondria have undergone (PT- or directly induced) MOMP but also to which extent mitochondrial damage has occurred or whether this process is still reversible.
  • PT- or directly induced mitochondrial populations
  • M3 up to four distinct mitochondrial populations (M0 to M3) emerge upon Ca 2+ -induced PT which can be detected by analyzing their electrophoretic mobility and/or surface charge, e.g.
  • zone electrophoresis in a free flow device and other suitable means to separate mitochondria by charge differences.
  • the relative amount and appearance of each of these populations is dependent either on the dose of the inducer of PT or the duration of induction.
  • a (quantitative) transition towards severely damaged organelles at higher doses or prolonged induction occurs in the form of a rise in the number of populations, a shift/transition of the populations towards the anode and/or an increase in the heterogeneity of one or more populations as compared to a reference sample.
  • An increased heterogeneity of one or more populations means that the variance within one population increases resulting, inter alia, in bigger differences in the surface charge or the electrophoretic mobility.
  • the examples disclosed in the present invention do not only show that in vitro induced PT/MOMP can be detected but also that MOMP directly arises upon ischemia/reperfusion. These results show that the findings in context of the present invention can be applied for research as well as for diagnostic purposes.
  • PT or PT-induced MOMP may also result in a more positive net surface charge as compared to unaffected mitochondria in a reference sample which is manifested in a transition or shift of the subpopulations towards the cathode.
  • these ions can either oxidize the negative charges on the exposed surface of the mitochondria or the ions themselves attached to the surface can cause the shift to a more positive net charge. This effect has to be distinguished from that expected upon a regeneration of the mitochondria.
  • a further method step has to be applied that serves to confirm that mitochondrial damage rather than an amelioration of the mitochondrial condition is present. Suitable means to confirm the status of the mitochondria or mitochondrial subpopulation(s), respectively, are described further below.
  • the detection of proteins usually not exposed on the outer surface of mitochondria by e.g. immunological means can be applied.
  • the present method provides means for the direct and quantitative analysis of the status of mitochondria.
  • the extent of mitochondrial damage detected is directly related to the number, the position and the heterogeneity of the subpopulations detected and can give rise to the status of the individual the sample was taken from or to a prognosis of this state.
  • the findings of the present invention and the method resulting herefrom can be applied in the analysis of disease states or conditions as will be described further below.
  • the present invention relates to a method of identifying an agent causing mitochondrial damage in mammalian cells comprising: (a) contacting with a test agent (i) a non-human animal and obtaining a sample containing tissue or cells to be analysed; or (ii) a sample obtained from a mammal and containing cells with mitochondria; or (iii) cultured cells containing mitochondria; or (iv) isolated mitochondria; and isolating the mitochondria from the sample of (i), (ii) or the cells of (iii); (b1) separating populations of the isolated mitochondria of step (a) by means of their electrophoretic mobility; and (c1) comparing the populations of the mitochondria obtained by analysing the electrophoretic mobility in (b1) with those of an untreated sample, wherein a shift of the population(s) towards the anode or the cathode, a rise in the number of mitochondrial populations and/or an increase in the heterogeneity of one or more populations separated in step (b1) as compared to the
  • an "untreated sample” as used in connection with the present invention can be a sample from the same source, i.e. from the same or a different tissue or organ of the same individual which has been obtained prior to the contacting step with the agent to be tested. In case of a different tissue or organ, it exerts a reaction in response to stimuli which is comparable to that of the tissues or organs the sample was taken from and/or have comparable properties as regards susceptibility to substances causing mitochondrial damage. If no such untreated sample is available, a suitable sample can also be obtained from a different individual, preferably of the same species, which was kept under the same conditions.
  • agents causing mitochondrial damage can directly or indirectly exert this effect.
  • Certain agents act directly on the mitochondria and cause PT or MOMP (e.g. viral proteins, reactive oxygen species (ROS), actractyloside, acetaminophen, ethanol, heavy metals; for a detailed list see Table 5S and 6S in Green and Kroemer, 2004), whereas others interfere with signal transduction pathways influencing mitochondria. Examples of such signal transduction pathways are apoptotic pathways or those resulting in an excess Ca 2+ concentration in the cell. Accordingly, in this aspect of the present invention, only agents directly damaging mitochondria can be detected using isolated mitochondria, whereas indirectly acting agents can be identified if samples containing cells or tissue are exposed to these agents in addition.
  • ROS reactive oxygen species
  • Contacting a non-human animal can be effected in various different ways. Nonlimiting examples are feeding, injection, application onto the skin or other parts of the body.
  • This second aspect of the present invention basically relies on the same finding and principles as described above for the method of detecting mitochondrial damage with a different purpose, i . e . the identification of agents causing mitochondrial damage.
  • the present invention relates to a method of identifying an agent capable of regenerating damaged mammalian mitochondria comprising: (a) contacting with a test agent (i) a non-human animal containing cells with damaged mitochondria and obtaining a sample containing tissue or cells to be analyzed; or (ii) a sample obtained from a mammal and containing cells with damaged mitochondria; or (iii) cultured cells containing damaged mitochondria; or (iv) isolated damaged mitochondria; and isolating the mitochondria from the sample of (i), (ii) or the cells of (iii); (b1) separating populations of the isolated mitochondria of step (a) by means of their electrophoretic mobility; and (c1) comparing the populations of the mitochondria obtained in (b1) by analysing the electrophoretic mobility with those of a reference sample not contacted with the test agent and containing damaged mitochondria, wherein a shift of the mitochondrial populations towards the cathode, a change in the distribution of the mitochondrial populations and/or a decrease in the heterogene
  • Capable of regenerating damaged mammalian mitochondria denotes the ability of an agent to reverse damage or ameliorate the state of mitochondria regarding their functional and structural intactness.
  • a reference sample as used in connection with this aspect of the present invention refers to a sample from the same ("the same source") or a different individual as the one contacted with the test agent.
  • the reference sample contains damaged mitochondria and has not been contacted with the agent to be tested. If necessary, the state of the mitochondria of the reference sample can be determined by comparison with those in another reference sample displaying a non-pathological phenotype.
  • the damage can, inter alia, be caused by ageing or stress, e.g. oxidative stress or intoxication with mitochondria damaging agents. If a sample from the same individual is used as a reference sample, it can be taken from the same tissue or organ as the sample to be measured or from a different tissue or organ with comparable properties as regards susceptibility to substances having regenerative capabilities.
  • the reference sample can be taken from a different individual.
  • the reference sample is preferably taken from the same tissue or organ as the sample to be analyzed.
  • the individual is preferably from the same species as that from which the sample to be measured was obtained.
  • This aspect of the present invention relies on the assumption that certain agents can have a regenerative effect on damaged mitochondria. Accordingly, by detecting differences in the total charges of mitochondria due to the additional exposure of inner charges, the method of the present invention can detect whether test agent have a regenerative effect on mitochondria.
  • the relative amount and appearance of each of the mitochondrial populations described in the context of the other aspects of the present invention is assumed to be dependent either on the dose of the potentially regenerative agent or the duration of exposure of the mitochondria or the sample analyzed to the agent.
  • a (quantitative) transition towards regenerated organelles at higher doses or prolonged exposure is assumed to occur in the form of a shift/transition of the populations towards the cathode, a change in the distribution of the mitochondrial populations and/or a decrease in the heterogeneity of one or more populations as compared to a reference sample.
  • a decreased heterogeneity of one or more populations means that the variance within one population decreases resulting, inter alia, in smaller differences in the surface charge or the electrophoretic mobility.
  • the profile obtained by analyzing the electrophoretic mobility of mitochondria by electrophoretic means and the breadth of the peaks observed therein directly indicates whether such a decrease in the heterogeneity has taken place.
  • a regeneration of mitochondria may also result in a more negative net surface charge as compared to unaffected mitochondria in a reference sample which is manifested in a transition or shift of the subpopulations towards the anode.
  • the mitochondria had been exposed to heavy metal ions followed by the exposure to an agent with regenerative capabilities, the more positive net charge caused by the heavy metal ions is counteracted through regeneration of the mitochondria.
  • the Applicant assumes that mechanisms exist causing the removal or masking of these positively charged ions. Accordingly, if a shift or transition of the mitochondrial subpopulations towards the anode is observed, a further method step has to be applied that serves to confirm that regeneration of mitochondria rather than mitochondrial damage is present. Suitable means to confirm the status of the mitochondria or mitochondrial subpopulation(s), respectively, are described further below.
  • the functional state of mitochondria may be assessed e . g . by respiratory measurements.
  • the methods of the present invention further comprise separating the mitochondria according to their electrophoretic mobility directly prior to the analysis of the average surface charge. Accordingly, the resulting populations are individually subjected to the analysis of the average surface charge.
  • the reference sample or untreated sample has to be treated accordingly, i.e. the mitochondria have to be separated according to their electrophoretic mobility prior to the analysis of the average surface charge in order to provide suitable means for comparison.
  • This additional method step will result in a clearer difference between the results obtained for the sample and those for the reference sample or the untreated sample since the average surface charge of isolated and thus homogenous subpopulations is analysed.
  • the separation according to the electrophoretic mobility is effected by free-flow electrophoresis.
  • Free flow electrophoresis is a process in which a sample stream is introduced into a liquid buffer flow within a separation vessel. A fixed or varying electric field is maintained across the separation vessel perpendicular to the buffer flow. Species of virtually identical biomolecules or bioparticles with different surface charges thus react or move differently within electric fields. Passing a selected sample of biomolecules or bioparticles through an electric field in an appropriate buffer or carrier results in the molecules migrating to separate positions or zones in the electrical field representative of their charge. Thus, visually and chemically similar biomolecules or bioparticles can be separated into subspecies according to their electrophoretic mobility. Individual components in the sample stream are separated from each other on the basis of their mobility in the imposed electric field and are collected at the exit of the separation vessel in one or several collection vessels (Hannig and Heidrich, 1990; Krivankova and Bocek, 1998).
  • the continuous zone electrophoresis (ZE-) FFE separation technique is based on the difference between the electrophoretic mobility of the particles to be separated.
  • ZE-FFE enables for the isolation of analytes and particles on the basis of differing size and/or shape and/or net surface charge.
  • the ZE-FFE method is especially suitable for separating sensitive bioparticles and complexes when specific demands have to be met by the separation medium during separation. This is particularly the case when the biological function and integrity of the particles and/or the original state of the particles prior to removal as a sample have to be maintained following separation.
  • the surface charge is analyzed by determining the zeta potential.
  • the zeta potential is the electric potential in the interfacial double layer at the slipping plane of a mobile particle in a suspension.
  • the electric potential describes the ability of an electric field caused by a charge to transfer power to other charges.
  • the interfacial double layer is a structure that appears on the surface of an object when it is placed into a liquid. This object may be a solid particle, gas bubble or liquid droplet.
  • the interfacial double layer is usually important when the size of the object is very small, on the scale of micrometers or even nanometers.
  • the structure of the interfacial double layer consists of two parallel layers of ions. One layer which is either positive or negative coincides with the surface of the object. The other layer is located in the fluid. This second layer electrically screens the first one.
  • Double layers exist in practically all heterogeneous fluid based systems, such as blood, protein solutions or suspensions containing mitochondria.
  • One class of phenomena arising from the existence of the interfacial double layer is electrokinetic phenomena.
  • electrokinetic phenomena the influence of an external force on the diffuse layer generates tangential motion of a fluid with respect to an adjacent charged surface. This force may be electric, a pressure gradient, a concentration gradient or gravity.
  • the moving phase may be either a continuous fluid or a dispersed phase.
  • Various combinations of the driving force and moving phase determine various electrokinetic effects.
  • the family of electrokinetic phenomena includes: electrophoresis (the motion of particles under the influence of an electric field); electro-osmosis (the motion of liquid in a porous body under the influence of an electric field); diffusiphoresis (the motion of particles under the influence of the chemical potential gradient); capillary osmosis (the motion of liquid in a porous body under the influence of the chemical potential gradient) and sedimentation potential (the electric field generated by sedimenting colloid particles).
  • the untreated or reference sample of step (c1) or (c2) and the sample, the cells or the mitochondria of step (a) are from the same source.
  • the term "same source" denotes that the sample and the untreated or reference sample are from the same individual.
  • the untreated or reference sample can either be taken from the same tissue or organ of the individual as the sample to be measured or from a different tissue or organ with comparable properties as regards susceptibility to substances having regenerative capabilities.
  • the method further comprises confirming that mitochondrial damage is present.
  • a confirmation step is necessary in the case that a shift of the population(s) to the cathode or a shift of the average surface charge to the positive is detected, such a step is not necessary if a shift of the population(s) to the anode or a shift of the average surface charge to the negative is detected since the latter result is expected in the majority of the examined cases.
  • a shift of the population(s) to the cathode or a shift of the average surface charge to the positive as observed e.g. upon exposure to heavy metal ions only constitutes a minority of cases.
  • the method further comprises confirming that the mitochondria have regenerated.
  • said method further comprises: (e1) comparing the electrophoretic mobility of the populations of the mitochondria obtained in (b1) with that of populations of a reference sample displaying a non-pathological phenotype, wherein a position, distribution and/or a heterogeneity of the mitochondrial populations similar to one or more of those of the reference sample is indicative of a regeneration of the mitochondria caused by the agent; or (e2) comparing the average surface charge determined in step (b2) with that of a reference sample displaying a non-pathological phenotype, wherein an average surface charge similar to that of the reference sample is indicative of a regeneration of the mitochondria caused by the agent.
  • Steps (e1) and (e2) can be carried out at any time after steps (b1) or (b2).
  • Step (e1) or (e2) serves to confirm the results obtained in steps (c1), (c2) or (d) by applying the approach that the electrochemical profile or surface charge of mitochondria which have regenerated should resemble those of a reference sample displaying a non-pathological phenotype.
  • This step is, however, not applicable if only a partial regeneration has taken place in the sample since in this case the profiles or surface charges of the sample and the reference sample displaying a non-pathological phenotype are not necessarily similar.
  • the present invention relates to a method of identifying an agent capable of regenerating damaged mammalian mitochondria comprising: (a) contacting with a test agent (i) a non-human animal containing cells with damaged mitochondria and obtaining a sample containing tissue or cells to be analyzed; or (ii) a sample obtained from a mammal and containing cells with damaged mitochondria; or (iii) cultured cells containing damaged mitochondria; or (iv) isolated damaged mitochondria; and isolating the mitochondria from the sample of (i), (ii) or the cells of (iii); (b1) separating populations of the isolated mitochondria of step (a) by means of their electrophoretic mobility; and (c1) (corresponding to step (e1) above) comparing the electrophoretic mobility of the populations of the mitochondria obtained in (b1) with that of populations of a reference sample displaying a non-pathological phenotype, wherein a position, distribution and/or a heterogeneity of the mitochondrial populations similar to one or more of those of the
  • reference sample displaying a non-pathological phenotype denotes a sample from the same or a different individual as the one from which the sample to be analyzed was taken. If a sample from the same individual is used as a reference sample, it can be taken from an undamaged part of the same tissue or organ as the sample to be measured or from a different tissue or organ with comparable properties as regards susceptibility to test agents. Alternatively, the reference sample can be taken from a different individual. In this regard, the reference sample is preferably taken from the same tissue or organ as the sample to which the test agent was applied. The individual is preferably from the same species as that from which the sample to be measured was obtained.
  • the mitochondrial damage is mitochondrial outer membrane permeabilization (MOMP).
  • MOMP mitochondrial outer membrane permeabilization
  • PT permeability transition
  • ANT adenine nucleotide translocator
  • the PT pore functions as a voltage sensor, as thiol sensor and as a sensor of Calcium-Ions: Matrix Ca 2+ ions increase, whereas external Ca 2+ ions decrease the probability of pore opening.
  • PT periodic reversible PT pore opening allows for the release of calcium from the mitochondrial matrix, thus allowing the maintenance of calcium homeostasis.
  • the opening of PT pores results in a sudden permeability increase of the inner mitochondrial membrane to solutes with a molecular weight of 1500 Da and some proteins, thereby disrupting the deltaPSI (the mitochondrial transmembrane potential) and associated mitochondrial functions.
  • deltaPSI the mitochondrial transmembrane potential
  • PT is accompanied by colloidosmotic swelling and uncoupling of oxidative phosphorylation, as well as by the loss of low molecular weight matrix molecules such as calcium and glutathione.
  • the concomitant extension of the mitochondrial inner membrane leads to a matrix transition from the "aggregated" to the "orthodox” state and finally results in MOMP.
  • the confirmation that mitochondrial damage caused by the agent or a regeneration of the mitochondria caused by the agent is present comprises determining the presence and optionally the quantity of proteins specific for the outer and/or inner mitochondrial membrane and/or for the inter membrane space in one or more of the populations.
  • the determination of proteins in this step of the method of the present invention describes a qualitative as well as a quantitative characterization of the proteins present on the outer and/or inner mitochondrial membrane and/or for the inter membrane space.
  • Examples for well-known assays based on qualitative and/or quantitative protein detection include without limitation method steps such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, high pressure liquid chromatography (HPLC), reversed phase HPLC, disc gel electrophoresis, two-dimensional gel electrophoresis, optionally in combination with mass spectrometric methods for protein identification, liquid chromatographic methods, optionally in combination with mass spectrometry, immunoblotting, immunoprecipitation, immune electrophoresis and enzymatic activity assays.
  • Typical references for these methods appear in specialized textbooks like the "Methods in Molecular Biology” or “Methods in Enzymology” series apart from single or review articles ( e . g . Rabilloud (2002)).
  • the mitochondrial damage detected results in cell death or regeneration/survival of the cells.
  • this overload can occur without damage to the mitochondria.
  • the overload is also accompanied by a combination of other factors, most notably oxidative stress, high phosphate concentrations and low adenine nucleotide concentrations, the mitochondria undergo PT leading to PT pore opening.
  • the resulting permeability includes protons, and thus the mitochondria become uncoupled. Since a pH gradient or membrane potential can no longer be maintained, the mitochondria not only become incapable of ATP synthesis, but also now actively degrade ATP.
  • Extracellular signals may include hormones, growth factors, nitric oxide or cytokines, and therefore must either cross the plasma membrane or transduce to effect a response. These signals may positively or negatively induce apoptosis; in this context the binding and subsequent initiation of apoptosis by a molecule is termed positive, whereas the active repression of apoptosis by a molecule is termed negative.
  • Intracellular apoptotic signalling is a response initiated by a cell in response to stress, and may ultimately result in cell suicide.
  • apoptotic signals Before the actual process of cell death is carried out by enzymes, apoptotic signals must be connected to the actual death pathway by way of regulatory proteins. This step allows apoptotic signals to either culminate in cell death, or be aborted should the cell no longer need to die resulting in survival or regeneration of the cell.
  • regulatory proteins Several proteins are involved, however two main methods of achieving regulation have been identified: targeting mitochondria functionality, or directly transducing the signal via adapter proteins to the apoptotic mechanisms. The whole preparation process requires energy and functioning cell machinery.
  • NO nitric oxide
  • SMACs second mitochondria-derived activator of caspases
  • SMAC binds to inhibitor of apoptosis proteins (IAPs) and deactivates them, preventing the IAPs from arresting the apoptotic process and therefore allowing apoptosis to proceed.
  • IAP also normally suppresses the activity of caspases, which carry out the degradation of the cell, therefore the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability.
  • Cytochrome c is also released from mitochondria due to increased permeability of the outer mitochondrial membrane, and serves as regulatory factor as it precedes morphological change associated with apoptosis.
  • cytochrome c Once cytochrome c is released it binds to Apaf-1 and ATP, which then bind to pro-caspase-9 to create a protein complex known as an apoptosome.
  • the apoptosome cleaves the pro-caspase to its active form of caspase-9, which in turn activates the effector caspase-3.
  • the mitochondrial permeability is itself subject to regulation by various proteins, such as those encoded by the mammalian Bcl-2 gene family of pro- and anti-apoptopic genes, the homologs of the ced-9 gene found in C. elegans.
  • Bcl-2 proteins are able to promote (e.g. BAX, BID, BAK or BAD) or inhibit (e.g.
  • Bcl-XI or Bcl-2) apoptosis by either direct action on mitochondrial permeability, or indirectly through other proteins.
  • the actions of some Bcl-2 proteins are able to halt apoptosis even if cytochrome c has been released by the mitochondria.
  • test agents capable of regenerating damaged mitochondria may act by increasing the expression and/or activity of these proteins.
  • apoptotic pathways utilize a multitude of different biochemical components, many of them not yet understood.
  • a pathway is more or less sequential in nature; removing or modifying one component leads to an effect in another.
  • the effects arising are often expressed in the form of a disease or disorder.
  • the concept overlying each disease or disorder is the same: the normal functioning of the pathway has been disrupted in such a way as to impair the ability of the cell to undergo normal apoptosis. This results in a cell which lives past its "use-by-date" and is able to replicate and pass on any faulty machinery to its progeny, increasing the likelihood of the cell becoming cancerous or diseased.
  • enhanced apoptosis can be the result of various non-cancerous diseases.
  • necrosis and apoptosis are interdependent phenomena resulting from activation of shared pathways and signals.
  • necrosis and apoptosis may be expected to occur in many pathophysiological settings.
  • the mitochondrial damage or condition of the mitochondria is indicative of a disease state or condition.
  • the disease state or condition is ischemia/reperfusion damage or injury, a neurodegenerative disease, a liver disease, cancer, a haematological disease or a viral infection.
  • Ischemia/reperfusion damage or injury refers to damage to tissue caused when blood supply returns to the tissue after a period of ischemia.
  • the absence of oxygen and nutrients from blood creates a condition in which the restoration of circulation results in inflammation and oxidative damage through the induction of oxidative stress rather than restoration of normal function.
  • the damage of reperfusion injury is due in part to the inflammatory response of damaged tissues.
  • White blood cells carried to the area by the newly returning blood release a host of inflammatory factors such as interleukins as well as free radicals in response to tissue damage.
  • the restored blood flow reintroduces oxygen within cells that damages cellular proteins, DNA, and the plasma membrane. Damage to the cell's membrane may in turn cause the release of more free radicals.
  • Such reactive species may also act indirectly in redox signaling to turn on apoptosis.
  • Leukocytes may also build up in small capillaries, obstructing them and leading to more ischemia.
  • Reperfusion injury plays a part in the brain's ischemic cascade, which is involved in stroke and brain trauma. Repeated bouts of ischemia and reperfusion injury also are thought to be a factor leading to the formation and failure to heal of chronic wounds such as pressure sores and diabetic foot ulcers. Continuous pressure limits the blood supply and causes ischemia, and the inflammation occurs during reperfusion. As this process is repeated, it eventually damages tissue enough to cause a wound.
  • AD Alzheimer's
  • PD Parkinson's
  • HD Huntington's
  • ALS amyotrophic lateral sclerosis
  • SMA spinal muscular atrophy
  • diabetic encephalopathy e.g. Alzheimer's (AD), Parkinson's (PD), Huntington's (HD) diseases, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and diabetic encephalopathy.
  • the traditional formulation of the amyloid hypothesis as a cause for AD points to the cytotoxicity of mature aggregated amyloid fibrils, which are believed to be the toxic form of the protein responsible for disrupting the cell's calcium ion homeostasis and thus inducing apoptosis.
  • liver disease is hepatic dysfunction which is a common clinical complication in malaria, although its pathogenesis remains largely unknown.
  • Malarial infection induces hepatic apoptosis through augmentation of oxidative stress.
  • Apoptosis furthermore plays a significant role in the course of hepatocyte death in acute and chronic hepatitis.
  • liver disease is acetaminophen hepatotoxicity (Masobuchi et al, 2005; reviewed in Malhi et al., 2006).
  • the hepatotoxicity of acetaminophen serves as an example of the interrelationship of necrotic and apoptotic cell killing and their common origin in mitochondrial dysfunction (see above).
  • acetaminophen overdose is the most frequent cause of acute drug-induced liver failure in the United States.
  • Cytochromes P450s metabolize acetaminophen to the reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI), which reacts with and depletes glutathione, forms covalent adducts and initiates mitochondrial oxidative stress. These events lead to onset of the MPT, and cyclosporin A protects against acetaminophen toxicity both in vitro and in vivo . As in other forms of liver injury, the roles played by oncotic necrosis and apoptosis in acetaminophen-induced liver damage have been controversial. Studies in mouse hepatocytes show that both modes of cell killing can predominate after acetaminophen exposure.
  • acetaminophen causes profound ATP depletion, ATP depletion-dependent necrotic cell killing ensues.
  • fructose and glycine are used to prevent ATP depletion, necrosis is blocked, whereas caspase-dependent apoptosis increases.
  • MPT mitochondrial inner membrane permeabilization
  • cyclosporin A decreases both the necrotic and the apoptotic modes of cell killing.
  • acetaminophen toxicity is one more example of necrapoptosis in which necrosis and apoptosis represent alternate outcomes of the same mitochondrial death pathway.
  • liver diseases are ischemia/reperfusion injury of the liver, death receptor injury and cholestatic liver injury (for a review see Malhi et al., 2006).
  • cancers are gastric cancer or hepatic cancer. It is known that cancer cells can be efficiently eradicated by apoptotic mechanisms. However, disruption of the apoptotic mechanism may cause the survival of cancer cells and the initiation of cell growth. Conventional anticancer therapies exert their effect by inducing apoptosis. However, a disrupted apoptotic pathway often impedes the efficiency of these therapies.
  • Hematological diseases related to apoptosis can be subdivided into those related to the loss of or escape from apoptosis and those related to excessive apoptosis.
  • apoptosis may occur in aplastic anemia, myelodysplastic syndromes (MDS), thalassemia, deficiency of hematopoietic factors such as in renal anemia or cytopenia due to anticancer agents or irradiation.
  • MDS myelodysplastic syndromes
  • thalassemia deficiency of hematopoietic factors
  • hematopoietic factors such as in renal anemia or cytopenia due to anticancer agents or irradiation.
  • Exemplary diseases and disorders caused by a loss or escape from apoptosis are haematological malignancies such as leukaemia, lymphoma or myeloma.
  • viruses encode proteins that can inhibit apoptosis.
  • viruses encode viral homologs of Bcl-2. These homologs can inhibit pro-apoptotic proteins such as BAX and BAK, which are essential for the activation of apoptosis.
  • pro-apoptotic proteins such as BAX and BAK, which are essential for the activation of apoptosis.
  • examples of viral Bcl-2 proteins include the Epstein-Barr virus BHRF1 protein and the adenovirus E1B 19K protein.
  • Some viruses express caspase inhibitors that inhibit caspase activity (e.g. the CrmA protein of cowpox viruses).
  • viruses can block the effects of TNF and Fas.
  • the M-T2 protein of myxoma viruses can bind TNF preventing it from binding the TNF receptor and inducing a response.
  • many viruses express p53 inhibitors that can bind p53 and inhibit its transcriptional transactivation activity. Consequently p53 cannot induce apoptosis since it cannot induce the expression of pro-apoptotic proteins.
  • the adenovirus E1B-55K protein and the hepatitis B virus HBx protein are examples of viral proteins that can perform such a function.
  • HIV human immunodeficiency virus
  • the method of the present invention further comprises analysing one or more of the obtained populations with one or more of electron microscopy, immunoblotting, immune electrophoresis, immunoprecipitation, cytofluorometric detection, HPLC, enzymatic activity assays, colocalization studies, detection of inner membrane surface metabolites, NMR, light scattering, atomic force microscopy, two-dimensional gel electrophoresis, optionally in combination with mass spectrometric methods for protein identification or liquid chromatographic methods, optionally in combination with mass spectrometry.
  • Typical references for these methods appear in specialized textbooks like the "Methods in Molecular Biology” or “Methods in Enzymology” series apart from single or review articles ( e . g . Rabilloud (2002)).
  • the test agent is a (cyto)toxic agent.
  • (Cyto)toxic agents as used in connection with the present invention denote agents which are directly toxic to cells. (Cyto)toxic agents causing apoptosis have been described elsewhere in this application. Well known assays to determine cytotoxicity of an agent are e . g . the colorimetric MTT, trypan blue, sulforhodamine assay and WST assays and the clonogenic assay.
  • a library of test agents is screened. Accordingly, the respective methods of the present invention are suitable for the use in high-throughput applications.
  • a library of test agents can comprise one class of molecules as well as a mixture thereof.
  • Suitable classes of molecules are, inter alia, peptides, nucleic acids, small molecules or aptamers. The general structure of these molecules is well-known in the art.
  • Peptides are a group of molecules consisting of up to 100 amino acids.
  • Nucleic acids comprise DNA, different types of RNA, such as RNAi, siRNA or shRNA as well as PNAs, a polyamide type of DNA analog.
  • RNAi RNAi
  • siRNA siRNA
  • PNA PNA
  • the monomeric units for the corresponding derivatives of adenine, guanine, thymine and cytosine are available commercially (for example from Perceptive Biosystems).
  • PNA is a synthetic DNA-mimic with an amide backbone in place of the sugar-phosphate backbone of DNA or RNA.
  • Aptamers are oligonucleic acid or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. Aptamers can be combined with ribozymes to self-cleave in the presence of their target molecule. These compound molecules have additional research, industrial and clinical applications.
  • a small molecule according to the present invention can be organic or inorganic and has a molecular weight of up to 2000 Daltons, preferably not more than 1000 Daltons, and most preferably not more than 800 Daltons.
  • High-throughput assays independently of being biochemical, cellular or other assays, generally may be performed in wells of microtiter plates, wherein each plate may contain 96, 384 or 1536 wells. Handling of the plates, including incubation at temperatures other than ambient temperature, and bringing into contact of test compounds with the assay mixture is preferably effected by one or more computer-controlled robotic systems including pipetting devices. In case large libraries of test compounds are to be screened and/or screening is to be effected within short time, mixtures of, for example 10, 20, 30, 40, 50 or 100 test compounds may be added to each well. In case a well exhibits biological activity, said mixture of test compounds may be de-convoluted to identify the one or more test compounds in said mixture giving rise to said activity.
  • Mitochondria were isolated by differential centrifugation according to standard protocols. Briefly, freshly removed liver tissue was cut into pieces and homogenized with a glass teflon homogenizer in isolation buffer (10 mM triethanolamine (TEA), 10 mM acetic acid (HAc), 280 mM sucrose, 0,2 mM EGTA, pH 7,4 with KOH). Homogenates were cleared from debris and nuclei by two times centrifugation at 750 g (10 min at 4°C) and mitochondria pelleted at 9000 g (10 min at 4°C).
  • TAA triethanolamine
  • Hc acetic acid
  • 280 mM sucrose 280 mM sucrose
  • 0,2 mM EGTA pH 7,4 with KOH
  • Organelles were washed three times (once at 9000 g and two times at 15000 g, 10 min, 4°C) and resuspended in FFE separation buffer (10 mM TEA, 10 mM HAc, 280 mM sucrose, pH 7.4 with KOH).
  • the arterial and portal blood flow to the left lateral and median lobe of the liver was interrupted by applying an atraumatic clip, resulting in a 70% liver ischemia. After 90 minutes of ischemia, the blood supply was restored by removal of the clip and the reperfusion period was initiated. Animals were sacrificed after 30 minutes or 3 hours of reperfusion, respectively, by bleeding. Control animals were sacrificed directly after the midline-laparotomy. The livers were rinsed free from blood by perfusing the organs with PBS.
  • Mitochondria were isolated from the left lateral and median lobe of the liver by differential centrifugation as above with the modification that mitochondria were pelleted from the 750 g supernatant at 15000 g (10 min at 4°C) and washed once at 15000 g. Mitochondria were stored in liquid nitrogen according to Fleischer (1979) until further use. For ZE-FFE experiments stored mitochondria were thawed, adapted to FFE separation buffer by washing and subsequently analyzed. For electron-scan microscopy, tissue samples were fixed in 3% glutaraldehyde for 2 hours and then kept in 1% glutaraldehyde until further examination. All animals received human care in compliance with the "Principles of Laboratory Animal Care". Studies were registered and approved by the government authorities.
  • IMVs were prepared from mitochondria by osmotic swelling-shrinking as described by Heidrich et al. (1970). PT-induced osmotic swelling of freshly isolated rat liver mitochondria suspensions upon Ca 2+ -addition was routinely measured by light scattering at 540 nm in a microplate absorbance reader ( ⁇ -QuantTM, Bio-Tek, Bad Friedrichshall, Germany) over a period of 30 min at RT. The final assay volume was 200 ⁇ l, containing mitochondria at 0.5 mg/ml in "standard swelling buffer" (10 mM MOPS-Tris pH 7.4, 200 mM sucrose, 5 mM succinate, 1 mM P i , 10 ⁇ M EGTA and 2 ⁇ M rotenone).
  • standard swelling buffer (10 mM MOPS-Tris pH 7.4, 200 mM sucrose, 5 mM succinate, 1 mM P i , 10 ⁇ M EGTA and 2 ⁇ M rotenone).
  • CsA-inhibited mitochondria were treated with CsA (0.6-4 ⁇ M) for 5 min and Ca 2+ (25 nmol/mg mitochondria) was subsequently added with 10 min further incubation.
  • Ca 2+ -treated mitochondria were incubated with different Ca 2+ -doses or reaction times as indicated in the text. Treated mitochondria were softly homogenized to ensure proper mixing, pelleted (15.000g, 10min, 4°C), re-suspended and analyzed by ZE-FFE. Untreated reference mitochondria were incubated in FFE-compatible swelling buffer alone, pelleted after 5 min and analyzed by ZE-FFE.
  • the mitochondrial samples suspended in FFE separation buffer were analyzed with the aid of a Free-Flow Electrophoresis apparatus (FFE-Weber GmbH, Planegg, Germany) with the following conditions: (i) circuit electrolyte solution was 100 mM HAc, 100 mM TEA adjusted to pH 7.4 with KOH; electrolyte stabilizing solution was 100 mM HAc, 100 mM TEA, 0.28 M sucrose, pH 7.4, FFE separation buffer was 10 mM TEA, 10 mM HAc, 280 mM sucrose, pH 7.4; (ii) all buffers were cooled on ice during separation to avoid thermal gradients; (iii) mitochondrial samples were applied to ZE-FFE with a sample flow rate of 1-2 ml/h; (iv) electrophoresis was performed in horizontal mode at 5°C with a flow rate of 330 ml/h and a voltage of 750 V; (v) fractions were collected in 96-well plates and the
  • ZE-FFE-separated mitochondrial fractions were immediately pelleted, fixed in 3% glutaraldehyde, post fixed with 1% osmium tetroxide, dehydrated with ethanol and embedded in Epon. Ultra-thin sections were negative stained with uranyl acetate and lead citrate and then analyzed on a Zeiss EM 10 CR electron microscope. Glutaraldehyde fixed liver tissues were treated similar but dehydrated with acetone and analyzed on a Philips EM 420 electron microscope.
  • Example 2 Discrimination of intact rat liver mitochondria and inner membrane vesicles by ZE-FFE.
  • Mitochondria have an "envelope-like" outer membrane and a structured inner membrane (Mannella, 2006). Permeabilities dramatically differ between these membranes, the outer membrane being freely permeable to low molecular weight solutes like sucrose, and the inner membrane being sucrose-impermeable. Incubation of purified mitochondria in low-osmolar or high-osmolar sucrose solutions leads to matrix water uptake and osmotic swelling or shrinkage, respectively. Such osmotic treatments rupture the mitochondrial outer membranes and generate inner membrane-matrix vesicles (IMVs) that can be electrophoretically distinguished from untreated mitochondria (Heidrich et al., 1970).
  • IMVs inner membrane-matrix vesicles
  • Example 3 Preparative analysis of mitochondria undergoing PT by ZE-FFE.
  • PT is typically monitored as a decrease in light scattering caused by mitochondrial matrix swelling (Beatrice et al., 1980) ( Fig 2A ). Due to its limited extensibility, the outer membrane ruptures upon matrix swelling (Van der Heiden et al., 1997).
  • Isolated rat liver mitochondria were energized with succinate, challenged for 20 min at RT with Ca 2+ (25 nmol/mg mitochondria) and subsequently analyzed by ZE-FFE ( Fig 2B ).
  • Energized mitochondria from the same preparation but without Ca 2+ -addition (“reference mitochondria”) were chosen to assess and compare the ZE-FFE deflection of undamaged mitochondria.
  • Energized mitochondria that were pre-treated with CsA before Ca 2+ addition (“CsA-inhibited mitochondria") were used as PT-inhibited control.
  • Electron microscopy revealed a morphologically heterogeneous mixture of mitochondria collected from M1 peaks ( Fig 2C , III), comprising mitochondria resembling intact reference mitochondria as well as a large portion of mitochondria which possessed dilated matrices and disorganised cristae ( Fig 2C , V and VI). M1 mitochondria were still surrounded by an outer membrane which was, however, damaged to different extents ( Fig 2C , III and below). In comparison to M1, M2 mitochondria were homogeneous vesicles with IMV morphology ( Fig 2C , IV and VII) and little outer membrane was detected in M2 mitochondria ( Fig 2C ).
  • Example 4 Ca 2+ -induced PT causes variable degrees of mitochondrial damage in a dose- and time-dependent fashion.
  • Energized mitochondria were incubated for 20 min at RT with Ca 2+ -doses ranging from 12.5 nmol Ca 2+ /mg mitochondria to 50 nmol Ca 2+ /mg mitochondria and subsequently analyzed by ZE-FFE ( Fig 3A ).
  • Fig 3A ZE-FFE separation profile
  • ZE-FFE is suitable for the quantitative and qualitative assessment of mitochondrial damage, as it is inflicted by Ca 2+ -induced PT in a dose- and time-dependent fashion.
  • Example 5 PT-induced MOMP in ischemia/reperfusion analyzed by ZE-FFE.
  • PT has been considered as an in vitro artefact for a long time (Bernardi et al., 2006), and many unsolved questions surround the role of the mitochondrial PT in vivo. It is generally accepted that mitochondrial membrane permeabilization represents a committing step in cell death scenarios (Green and Kroemer, 2004). It is a matter of controversy, however, whether PT is a cause or a consequence of cell death (Kinally and Antonsson, 2007), under which conditions and to which extent PT occurs in vivo (Bernardi et al., 2006), and which threshold of PT is associated with cell death in distinct cell types (Rodriguez-Enriquez et al., 2004).
  • Ischemia leads to mitochondrial de-energization, ATP depletion, lactic acid accumulation and consequent intracellular pH drop. Consequently cells are loaded with Ca 2+ due to the action of the Na + /H + and Na + /Ca 2+ -antiporters.
  • pH normalizes, mitochondria re-energize and take up Ca 2+ , setting the conditions for PT to occur (Halestrap, 2006).
  • I/R did not induce the appearance of vesicles with IMV morphology that would resemble M2 mitochondria ( FIG 2C , VII), suggesting that a full-blown PT did not occur under these conditions. Rather, I/R-damaged mitochondria morphologically resembled M1 mitochondria (compare Fig 4C , II and IV with Fig 2C , III, V and VI).
  • Example 6 Confirmation of different surface potentials by determining the zeta potential.
  • both electrophoretic methods are sensitive to the altered surface potential of PT-damaged mitochondria in comparison to control organelles.
  • this result is to be expected, since both methods are sensitive to the electrophoretic mobility of the investigated particles, on the other hand, however, this result shows that it is the mitochondrial damage/difference which is detected independently of the method used to assess it. Consequently both methods may be employed to detect mitochondrial damage, thus enabling the use of the appropriate technique depending on the experimental circumstances.
  • the Zetasizer appears to have clear advantages regarding low amounts of analysis material (e . g . originating from biopsies or cell culture) and low analysis time.
  • the electrophoretic free flow approach is a preparative method by which the separated mitochondria can be collected and subsequently analyzed by biochemical or functional methods. Furthermore several subpopulations emerging from damaging conditions may be separated by this latter approach, thus resulting in a more detailed analytical picture.
  • Irreversible outer mitochondrial membrane damage turns the cellular switch towards death and several approaches aim to monitor this "point of no return".
  • This approach provides an analytical tool and, more importantly, a preparative method for the purification of mitochondria from higher eukaryotes that have undergone distinct level of damage in vitro or in vivo.
  • Table 1 Samples 1 to 4 measured on July 12 2007 Samples 1 to 4 were measured in their original medium measurements conducted on the Zetasizer Nano ZS device with the standard sample cell DTS1060 Record Type Sample Name Measurement Date and Time T °C ZP mV Mob ⁇ mcm/Vs Cond mS/cm Current mA Voltage V Quality Zeta Runs 1 Zeta sample 1 FIII 02.07.07 control 1 Friday, July 13 2007 15:05:10 25 -12,4 -0,9687 0,654 0,253 30 1,76 12 2 Zeta sample 1 FIII 02.07.07 control 2 Friday, July 13 2007 15:06:11 25 -11,5 -0,9036 0,655 0,253 30 1,46 12 3 Zeta sample 1 FIII 02.07.07 control 3 Friday, July 13 2007 15:07:26 25 -13,2 -1,031 0,656 0,253 30 1,59 12 4 Zeta sample 1 FIII 02.07.07 control 4 Friday, July 13 2007 15:08:41 25
  • ZE-FFE can be employed for the discrimination and characterization of mitochondria exhibiting increasing levels of MOMP.
  • MOMP metal-oxide-semiconductor
  • M2 major stage of PT
  • M1 intermediate stage
  • PT-associated MOMP may progress through discrete steps. It will be interesting to see whether further mitochondrial subpopulations emerge, e . g . when PT is induced by other triggers.
  • ZE-FFE will provide a welcome addition to the methodological armamentarium of mitochondrial research.
  • ZE-FFE is a novel tool for analyzing and purifying mitochondria that have undergone defined levels of damage.

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